Calcineurin-dependent regulation of renal Na-(K-)Cl-cotransporters (II)

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Regulation of salt and water homeostasis and blood pressure critically depends on tubular epithelial functions in the kidney. The distal Nephron (thick ascending limb [TAL], distal convoluted tubule [DCT]) determines volume regulation effectively via salt reabsorption. The kidney-specific Na-(K-)Cl-cotransporters (cation-chloride cotransporters, CCC) comprise NKCC2 in TAL and NCC in DCT. Both are activated by a cascade of phosphokinases (WNK/SPAK/OSR1). Defects in the cascade cause alterations in homeostasis. In monogenetic pseudohypoaldosteronism (PHA-II) with tubular salt retention we have characterized WNK1 and WNK4 functions, established a link to their cullin/kelch-like-mediated degradation, and described a new, overactivating WNK1 mutant. The calcium-dependent serine-theorine phosphatase calcineurin controls these activating steps. Calcineurin Inhibitors (CNI; tacrolimus, cyclosporin A) are successfully administered for immunosuppression after transplantation, but side effects such as volume retention and hypertension are common. We have shown that CNI may in part phenocopy PHA-II with tubular salt retention, which could be reduced by inhibiting NCC with thiazide diuretics. An involvement of NKCC2 was demonstrated as well. To analyze the mechanisms involved, calcineurin isoforms and CNI-binding immunophilins were localized, and an immunophilin-deficient mouse model evaluated. Analysis of the binding properties between calcineurin, the kinases, and the CCC led us to the identification of essential, non-redundant mediator proteins such as annexin A2 for the trafficking and lipid-raft association of NKCC2, as well as calcineurin homologous protein 1 (CHP1) and SORLA to transmit calcineurin effects on NKCC2. During the renewal period, we will follow the hypothesis that calcineurin differntially exerts important regulatory functions in TAL as well as in DCT. The interactions between CCC, calcineurin, and kinases will be validated mechanistically. The phosphatase activity of calcineurin will be determined and specified at the molecular level. The role of scaffold proteins as well as the impact of annexin A2, CHP1 and SORLA will be verified in the context of CCC phosphoregulation. Proteasomal degradation, aggresome formation and autophagy will be studied during adaptive restructuring of DCT upon furosemide therapy and diet-induced transport stimulation, considering an involvement of calcineurin and WNK-1 functions. Volume retention caused by a high-salt, low-potassium diet ("Western diet") will be analyzed with respect to regulatory adaptations of the WNK-SPAK/OSR1-CN-CCC cascade. Sustained CNI effects will be studied to register status and interactions of the identified products, and endocrine effects will be considered. Therapeutic options addressing the side effects of immunosuppressive therapy shall be achieved based on the results of this project.